Art
[1]
The present invention relates to a raised novel Lactobacillus bariwooseu CJLS1511, animal feed additive composition, and method for producing the four cells comprising the fungus or its four cells.
[2]
BACKGROUND
[3]
Lactobacillus genus (Lactobacillus sp.) Microorganisms are often found in the fermentation process of the Minister and dairy products and vegetables of animals, including humans as a lactic acid bacillus that a homo- or hetero-fermentation. Lactobacillus genus microorganism is a lactic acid producing bacteria that are found in the views of the animal, using a dairy or vegetable as a counter substrate and a homogeneous fermentation (homo-fermentation) or heterogeneous fermentation (hetero-fermentation). Lactobacillus bacteria in the microorganism to maintain the pH in the intestines acid, Escherichia coli (E. coli) and Clostridium (Clostridium) inhibit the growth of harmful bacteria as well as to improve diarrhea and constipation, vitamin synthesis, anticancer action, such as, serum It is known to play a role, such as lowering cholesterol.
[4]
Lactobacillus has been actively conducted research to develop a probiotic and animal feed with the properties of the Bacillus microbe. Bacterial diarrhea in livestock leads to reduced growth rate and mortality. Therefore, this prevention have been made to generally as well to increase the production of livestock chair addition of antibiotics in feeds. However, restricting the use of antibiotics in food because of problems such as emergence and livestock residual antibiotics resistant to antibiotics and is a trend that contains organic animal specification method is emphasized (Republic of Korea Patent Application Publication No. 1998-78358 No.) (McEwen and Fedorka-Cray, Antimicrobial use and resistance in animals, Clinical infectious Diseases, Volume 34, June 2002, pages S93-S106).
[5]
Detailed Description of the Invention
SUMMARY
[6]
Thus, in one aspect of the present invention, it is possible to improve the weight gain in using animals with the animal feed, it improves the immune system or hangbyeongryeok animal, new capable of inhibiting the enteropathogenic bacterial growth of the animal a Lactobacillus genus (Lactobacillus sp.) to provide a microorganism.
[7]
Problem solving means
[8]
One aspect of the invention, Lactobacillus salivarius CJLS1511 ( Lactobacillus salivarius relates to four cells CJLS1511) (KCCM11829P) or derivatives.
[9]
Other aspects of the present invention relates to Lactobacillus salivarius CJLS1511 (KCCM11829P) or animal feed additive composition comprising a counter four cells.
[10]
Another aspect of the invention relates to animal feed comprising an animal feed additive composition described herein.
[11]
Yet another aspect of the present invention,
[12]
Lactobacillus raised by culturing bariwooseu CJLS1511 (KCCM11829P) preparing a culture medium,
[13]
The culture medium is heated at a temperature of 70 ℃ to 160 ℃,
[14]
The heated culture solution, cooled to a temperature in the range of 10 ℃ to 60 ℃,
[15]
, Lactobacillus raised it relates to a process for producing bariwooseu CJLS1511 (KCCM11829P) four cells, comprising separating the Lactobacillus raised bariwooseu CJLS1511 (KCCM11829P) cells were used in the cooling medium.
[16]
Effects of the Invention
[17]
Lactobacillus salivarius CJLS1511 or a four cell according to one aspect of the present invention is excellent in resolution triglycerides, endotoxin adsorption capacity, pathogenic bacteria growth inhibitory effect and digestive enzymes bow performance.
[18]
Lactobacillus salivarius animal feed additive or animal feed composition comprising a CJLS1511 or a four cell according to one aspect of the present invention can improve the weight gain of animals and improving immunity to hangbyeongryeok.
[19]
Brief Description of the Drawings
[20]
1 is Lactobacillus salivarius ( Lactobacillus salivarius shows an electron micrograph of a) CJLS1511 strain or its four cells.
[21]
Figure 2 shows a phylogenetic tree of the salicylate Lactobacillus bariwooseu CJLS1511 (phylogenetic tree).
[22]
Mode for the Invention
[23]
It will now be described in more detail with respect to the present invention. Details are not described herein because it can be sufficiently recognized and deduced if one skilled in the art or the similar aspect of the present invention and the description thereof will be omitted.
[24]
One aspect of the invention, Lactobacillus salivarius CJLS1511 ( Lactobacillus salivarius is for the four cells CJLS1511) (KCCM11829P) or derivatives.
[25]
Herein, Lactobacillus salivarius ( Lactobacillus salivarius use of) CJLS1511) (KCCM11829P) cells "include, for example, Lactobacillus salivarius ( Lactobacillus salivarius inactive cells of) CJLS1511) (KCCM11829P), specifically, the deactivated by heat cells can be.
[26]
The inventors have anaerobic incubation at 37 ℃ to spread on the MRS medium was added, and then the purple 0.001% boric penny call (Bromphenicol purple, BCP) and washed with sterile distilled water to collect the small intestine ends of the broilers. Wherein the sub-cultured to screen for more than 50 kinds of lactic acid producing strains were isolated following the second 24 species of bacillus form morphologically separation method using the strain. Antimicrobial activity and digestive enzymes of 10 kinds were isolated tertiary compares active power, in bile, acid resistance and animal barrier measuring cell adsorption, and fermented sugar, pathogenic bacteria growth inhibitory effect of the lactic acid producing strains for the 24 species, digestion the enzyme active performance, triglycerides resolution to separate the best one kind of strain.
[27]
Wherein the lactic acid bacteria produce strains Lactobacillus salivarius CJLS1511 ( Lactobacillus salivarius was named CJLS1511) and deposited to 412 of January 2016 to the Korea Culture Center of Microorganisms (accession number: KCCM11829P).
[28]
Morphological and physiological characteristics of the Lactobacillus salivarius CJLS1511 are shown in Table 1 below.
[29]
TABLE 1
1. Morphological characteristics (in the form of sikyeoteul when grown in MRS solid medium)
- shaped cells Bacilli
- Polymorphism of cells (多 形 性) none
- mobility none
- forming Apo none
2. Physiological characteristics
- Gram stain (Gram staining) Gram-positive (Gram positive)
- catalase (Catalase) voice
- oxidase (Oxidase) voice
- the growth temperature and time 37 ° C, 18 ~ 48 sigan
Oxygen is also required Facultative anaerobic

[30]
[31]
Also, the Lactobacillus salivarius CJLS1511 does not form spores has the form bar (rod), as shown in FIG. By the activity per protein of the cell wall surface when the disproportionated four strains is verified by the difference between live cells and dead cells.
[32]
The Lactobacillus bacteria alive for a biochemical analysis of bariwooseu CJLS1511 analyzed by API kit, to raise Lactobacillus bacteria as shown in Table 2 bariwooseu ATCC11741 or KCCM 40210 were identified as having a utilization per similar to the type strain 16s rRNA analysis to Macrogen request by Lactobacillus salivarius ATCC11741 ( Lactobacillus salivarius has also been identified, such as 2 to have ATCC11741) or KCCM 40210 type strain and 99% similar molecular properties.
[33]
Table 2. Lactobacillus raised using feature analysis per bariwooseu CJLS1511
No. Per type Lactobacillus salivarius CJLS1511
0 Witness -
1 Glycerol -
2 Erythritol -
3 D-arabinose -
4 L-arabinose -
5 D-ribose -
6 D-xylose -
7 L-xylose -
8 D-adonito -
9 Methyl-ａD-xylopyranozide +
10 D-galactose +
11 D-glucose +
12 D-fructose +
13 D-mannose -
14 L-sorbose -
15 L-ramnose -
16 D-ulcitol -
17 Inositol +
18 D-mannitol +
19 D-sorbitol +
20 Methyl-Ad-manopyranozide -
21 Methyl-ａD-glucopyranozide -
22 N-acetylglucosamine +
23 Amygdalin -
24 Arbutin +
25 Asculin -
26 Salicin +
27 D-celobiose -
28 D-maltose +
29 D-lactose +
30 D-mellibiose +
31 D-saccharose +
32 D-trehalose +
33 Inulin -
34 D-melesitose -
35 D-rafinose +
36 Starch -
37 Glycogen -
38 Xylitol -
39 Genthiobiose -
40 D-turanose -
41 D-lyxose -
42 D-tagatose -
43 D-fucose -
44 L-fucose -
45 D-arabitol -
46 L-arabitol -
47 Gluconate -
48 2-ketogluconate -
49 5-ketogluconate -

[34]
The Lactobacillus salivarius CJLS1511 (KCCM11829P) are the acid resistance, within the biliary, antimicrobial activity, digestive enzymes bow performance and triglycerides resolution is excellent it can improve the efficiency and weight gain of the animal when using the feed in the animal feed or feed composition effectively there is an advantage.
[35]
The Lactobacillus salivarius CJLS1511 (KCCM11829P) four cells could contribute to form the intestinal flora to achieve the enteropathogenic bacteria and competitive adhesion inhibition, reports of the cell walls of the four cells of the eluted cell wall configuration while destroying component in the small intestine table of the Kosan (lipoteichoic acid) is enteropathogenic bacteria may prevent settling in intestinal mucosa. There also is an advantage that can raise the four cells of Lactobacillus bariwooseu CJLS1511 (KCCM11829P) is hydrophobic and cohesion higher than the live cells attached to the pathogenic microorganisms and endotoxin improves hangbyeongryeok. Moreover, the Lactobacillus bacteria raised by administering the four cells of bariwooseu CJLS1511 (KCCM11829P) to feed if the class in animals can be improved both weight gain and feed conversion.
[36]
Lactobacillus salivarius CJLS1511 (KCCM11829P) or a four cell may further include a protective agent. Examples of the protective agent may be more than one kinds of seoltangryu such as yeast, yeast extract, monosaccharides, polysaccharides, starches, sugar and jeongjedang. Specifically, there may be mentioned one kind of substance selected from the group consisting of yeast extract, dextrose and sugar. It is possible by using the protective agent Lactobacillus raised to protect bariwooseu CJLS1511 (KCCM11829P) or a four cell from the external environment to prevent contamination, it is effective to extend the shelf life thereof.
[37]
Another embodiment of the invention, is for the animal feed additive composition of the Lactobacillus bacteria alive including bariwooseu CJLS1511 (KCCM11829P) or a four cell. Specifically, the animal feed additive composition may raise Lactobacillus include four cells of bariwooseu CJLS1511 (KCCM11829P).
[38]
The animal feed additive composition may further comprise an excipient. The vehicle shall not be particularly limited and may be an excipient that conventionally used in the art. As the excipients, for example sugar (e.g., lactose, D- mannitol, D- sorbitol, sucrose, etc.), gums (such as xanthan gum, guar gum, gum arabic sangeom, etc.), starches (e.g., corn starch, potato starch, etc.), inorganic and the like: (calcium phosphate, calcium sulfate, precipitated calcium carbonate, etc.) salts.
[39]
The animal feed additive composition, raise the Lactobacillus bacteria per 1g composition bariwooseu CJLS1511 (KCCM11829P) or the use thereof, the cells 1.0x10 8 cfu to 1.0x10 10 may be included as cfu. Specifically, 1.0x10 8 cfu to 3.0x10 9 may be one comprising cfu, 5x10 In one example 8 may include a cfu.
[40]
The animal feed additive compositions may be in the form of a powder, pellets, or granules. Animal feed additive may be a composition of the Lactobacillus salivarius CJLS1511 (KCCM11829P) or content of use thereof, the cells are in the range of 0.1% to 10% by weight, in particular in the range of 0.1% to 7% by weight. When the composition in the range including the strain or its four cells, there is an advantage in that the animal feed additive of the Resolution triglyceride composition, endotoxin adsorption capacity, pathogenic bacteria growth inhibitory ability and digestive enzymes bow performance can be maximized.
[41]
The cost of animal feed prepared in a further aspect, using the animal feed additive composition is provided. The animal feed may be one prepared in combination with an animal feed additive composition, conventional animal feed in accordance with one aspect of the present invention. The amount present in the animal feed additive of animal feed composition may be 0.1% to 1% by weight based on the weight of the animal feed.
[42]
Animal feed may be not particularly limited, and feed for farm animals. It is meant to include "animal husbandry" means milk, meat, eggs, wool, leather, animal and pets for the production of useful livestock and fisheries to human beings, such as feathers. As specific examples, there can be for a class breeding animals such as dogs, cows, chickens, pigs, and horses. More specifically, poultry, such as chicken, can be to the level in the broiler in more detail.
[43]
Animal feed may comprise an integral component of the carbohydrate, protein, fat, vitamins and minerals for growth of the animal. Protein may be an animal protein or vegetable protein, for example, six minutes, poultry minutes, fish meal, and the like soy protein, milk protein, or gluten. As the carbohydrate, there may be mentioned cereals, or legumes, e.g., corn, rice, wheat, barley, oats, soy, or mixtures thereof. The lipid in the lipid may be an animal, vegetable or meat lipids. In addition to the above components or it may further comprise other components that add functionality to the animal feed. Or, it may be mixed with sugar, salt, spices, seasonings, and flavors.
[44]
Another embodiment of the invention, Lactobacillus raised by culturing bariwooseu CJLS1511 (KCCM11829P) preparing a culture medium,
[45]
The culture medium is heated at a temperature of 70 ℃ to 160 ℃,
[46]
The heated culture solution, cooled to a temperature in the range of 10 ℃ to 60 ℃,
[47]
, Lactobacillus raised it relates to a process for producing bariwooseu CJLS1511 (KCCM11829P) four cells, comprising separating the Lactobacillus raised bariwooseu CJLS1511 (KCCM11829P) cells were used in the cooling medium.
[48]
More specifically, the method comprising the Lactobacillus raised by culturing bariwooseu CJLS1511 (KCCM11829P) preparing a culture medium may be an agar medium, specifically, MRS medium. The incubation was at 25 ℃ to 40 ℃ 5 hours to about 48 hours, specifically, 30 ℃ to from 40 ℃ 12 hour to 36 hour incubation, more specifically from 35 ℃ to from 40 ℃ 20 hour to 30 hour incubation with the culture medium the can be prepared.
[49]
Then, the culture medium is heated to the culture medium at a temperature of 70 ℃ to 160 ℃. The heating, but can take advantage of a direct or indirect heating means, the heating means, can be via the indirect heating means such as a heat exchanger. The heating is in particular 80 ℃ to 150 ℃, more specifically, can be carried out at a temperature ranging from 90 ℃ to 120 ℃. The bariwooseu CJLS1511 raised Lactobacillus through the heating may hwahal inactivated or dead cells.
[50]
In the heating after the culture medium 10 to 60 ℃, specifically cooled to a temperature of 20 to 50 ℃ or, one example can be rapidly cooled to 4 ℃. The cooling is 10 ℃ / min to 60 ℃ / min, specifically, may be cooled at a rate of 20 ℃ / min to 50 ℃ / min, more specifically 30 ℃ / min to 40 ℃ / min. Then, by separating the four cells in the cooled broth Lactobacillus salicylate it can be obtained bariwooseu CJLS1511 (KCCM11829P) cells were used.
[51]
The Lactobacillus salivarius CJLS1511 (KCCM11829P) preparation of four cells may include, in addition to blend the protective agent in the separated four cells. Further, since it is possible to further comprises a powdered mixture of the four cells with the agent. Mixing a protective agent as described above to prevent the contamination from by triturated this mixture, cells were used for an external environment, and there is an advantage that can facilitate the distribution of the four cells.
[52]
The protective agent is one or not is not particularly limited, for example, can be more than one kinds of seoltangryu such as yeast, yeast extract, monosaccharides, polysaccharides, starches, sugar and jeongjedang. Specifically, it is possible to use one kind of substance selected from yeast extract, dextrose, and the group consisting of sugar.
[53]
The pulverization may be used for freeze-drying, spray drying or spray agglomeration, more specifically may utilize spray drying. Also referred to as spray drying is spray drying, the method instantaneously obtain a dry product of liquid phase at once spraying a liquid during yeolgiryu, as a method of liquid to the spray shape, the centrifugal atomization and pressure spraying by a pressure nozzle according to the rotation disc. In order to conduct the spray-dried skimmed milk or the like, it is possible to use spray-drying apparatus well known those employing a spraying method.
[54]
Considering the ease of operation created by the mixture was mixed for four cells and a protective agent and then, after suspending the mixture in water, to the spray-drying process the suspension is obtained, that is to 120 ~ 200 ℃ the inlet temperature of hot air, preferably to 130 ~ 170 ℃ and it is preferred to spray-drying to the outlet temperature of 30 ~ 150 ℃, preferably 50 ~ 100 ℃. The amount of protective agent is in parts by weight based on 100 parts by weight of the four cells, 0.1 parts by weight to 300 parts by weight, specifically, may be an 0.1 parts by weight to 200 parts by weight.
[55]
In one example, the added amount of yeast or yeast extract is 0.04 to 50 parts by weight, preferably from 0.1 to 10 parts by weight On the other hand, the addition amount of the monosaccharides 1 and 100 parts by weight, preferably from parts 10 to 50 by weight, and the seoltangryu that and the addition amount 0.2 to 50 parts by weight, preferably 0.4 parts to 10 parts by weight.
[56]
When used as a pulverization operation, in particular additives for inorganic physiologically acceptable, such as adult calcium sparingly soluble in water in the spray-drying operation was confirmed that the operation become easy. In this case, 1 to 99% by weight of the amount of the additive composition of the inorganic material, specifically, more specifically, 1 to 90% by weight, from 1 to 10% by weight. In one example, the additive composition used cell powder 0.05 to 50% by weight, preferably from 0.5 to 20% by weight, more preferably it comprises 0.5 to 15% by weight, the inorganic material 5 to 80% by weight, preferably from 5 to 50%, more preferably containing 5 to 20%.
[57]
Cells were used in the additive composition will raise the Lactobacillus bacteria per 1g the composition bariwooseu CJLS1511 (KCCM11829P) or a four cell is 1.0x10 8 cfu to 1.0x10 10 may be included as cfu. Specifically, 1.0x10 8 cfu to 3.0x10 9 may be one comprising cfu, 5x10 In one example 8 may include a cfu.
[58]
[59]
Or less, with a preferred embodiment of the present invention will be described in more detail the construction and operation of the present invention. However, it is not in any sense will set forth a preferred example of the present invention can also be construed as limiting the invention thereby.
[60]
Details are not described herein, if one skilled in the art because it can be sufficiently inferred technically will be omitted.
[61]
[62]
Example
[63]
Test Example 1-6
[64]
The known strains Lactobacillus raised by the bariwooseu KCCM 40210 in comparison strain, salicylate Lactobacillus of the present invention follows the bariwooseu CJLS1511 (KCCM11829P) safety, acid resistance, within the biliary, antimicrobial activity, digestive enzyme activity and the triglyceride degradation activity of the strain It was evaluated as.
[65]
[66]
Experimental Example 1: Lactobacillus salivarius of CJLS1511 (KCCM11829P) Safety Assessment
[67]
Lactobacillus salivarius CJLS1511 (KCCM11829P) to assess the safety of strains of hemolysis, depending on the safety evaluation test method presented in Korea Bio Venture Association Associations phenomena test, gelatin liquefaction reaction test, toxic metabolites check (ammonia) generation and Peninsula alanine It was carried out for de-amine test. The results are shown in Table 3.
[68]
TABLE 3
Strain Item
　 Gelatin liquefaction reaction test Phenylalanine deionized amine test Hemolysis test development Ammonia generated
Lactobacillus salivarius CJLS1511 voice voice a- hemolysin, safety voice

[69]
As can be seen in Table 3, Lactobacillus salivarius CJLS1511 (KCCM11829P) is exhibited in both the negative gelatin liquefaction reaction check, confirmation tests Peninsula alanine deionized amine test, and ammonia generation, it turned out to be safe in the hemolysis test.
[70]
[71]
Experimental Example 2: Lactobacillus salivarius of CJLS1511 (KCCM11829P) acid resistance evaluation
[72]
Pre-cultured in MRS broth Lactobacillus raised bariwooseu raised CJLS1511 strains and comparing the strains Lactobacillus bariwooseu KCCM 40210 in MRS 10 solution was adjusted to each pH 2, 3, 7 for the type strain -6 diluted times. To smear the diluted bacterial solution on MRS agar medium by a constant time and incubated at 37 ℃ and incubated anaerobically for 48 hours to measure the number of colonies. Results of the acid resistance test are shown in Table 4.
[73]
TABLE 4
Strains MRS broth (pH7) MRS broth (pH2) MRS broth (pH3)
Live cell number (CFU / mL) Live cell number (CFU / mL) Live cell number (CFU / mL)
0 h 1 h 3 h 0 h 1 h 3 h 0 h 1 h 3 h
Lactobacillus salivarius CJLS1511 5.8 x 108 6.0 x 108 6.2 x 108 5.8 x 108 2.3 x 105 2.0 x 105 5.8 x 108 3.5 x 108 2.0 x 108
Lactobacillus salivarius KCCM 40210 6.4 x 108 6.8 x 108 8.6 x 108 6.4 x 108 2.0 x 102 2.0 x 102 6.4 x 108 1.0 x 108 5.0 x 106

[74]
As can be seen in Table 4, Lactobacillus raised bariwooseu CJLS1511 showed that fewer pH 2 to 4, taking advantage of Lactobacillus strains in comparison bariwooseu KCCM 40210 reduced the number of bacteria to survive than strains having excellent acid resistance.
[75]
[76]
Experimental Example 3: Lactobacillus salivarius of CJLS1511 (KCCM11829P) within the biliary evaluation
[77]
Pre-cultured Lactobacillus raised bariwooseu CJLS1511 strains and comparison strains of Lactobacillus raised after each adjusting bariwooseu KCCM 40210 type strain to pH 4, 0% to bile acid solution (Oxgall), 0.3% in MRS broth, concentration of 1% a, 10 a MRS solution Align -6 was diluted. To smear the diluted bacterial solution on MRS agar medium by a constant time and incubated at 37 ℃ and incubated anaerobically for 48 hours to measure the number of colonies. As a result of measuring the number of bacteria are summarized in Table 5.
[78]
Table 5
Strains MRS broth (pH4) MRS broth(pH4 and Oxgall 0.3%) MRS broth(pH4 and Oxgall 1%)
Live cell number (CFU / mL) Live cell number (CFU / mL) Live cell number (CFU / mL)
0 h 1 h 3 h 0 h 1 h 3 h 0 h 1 h 3 h
Lactobacillus salivarius CJLS1511 2.4 x 108 4 x 108 5.6 x 108 2.4 x 108 4.0 x 106 2.0 x 104 2.4 x 108 5.6 x 105 5.2 x 103
Lactobacillus salivarius KCCM 40210 2.0 x 108 2.0 x 108 2.2 x 108 2.0 x 108 2.6 x 105 2.0 x 102 2.0 x 108 2.0 x 102 2.0 x 102

[79]
As can be seen from Table 5, Lactobacillus raised bariwooseu CJLS1511 is pH 4, a bile acid (Oxgall) 0.3% and 1% solutions, but both a decrease in the number of surviving bacteria in, Lactobacillus raised reduction in bacterial counts degree than bariwooseu KCCM 40210 type strain is even much less bile acid secretion in an animal body, it was confirmed that the administration of the strain to grow.
[80]
[81]
Experimental Example 4: Lactobacillus salivarius of CJLS1511 (KCCM11829P) antibiotic activity evaluation
[82]
TSB (Tryptic soy broth) medium (BD, USA) 24 sigan liquid pathogens of the three kinds of the culture (in E. coli K88, E. coli ATCC 25922, Salmonella typhimurium KCCM 25922, Salmonella cholerasuis uniform in a sterile swab to KCCM 10709) 10 to 5-6 were plated in cfu / ml.
[83]
And then spread on the paper disc diameter 4mm Lactobacillus raised bariwooseu CJLS1511 and Lactobacillus raised bariwooseu KCCM 40210 type strain 10 in PBS buffer, respectively 9 and then diluted with cfu / mL was dispensed by 50㎕. The diluted solution was incubated aerobically for 18 hours from the fully left at room temperature until the next 37 ℃ permeate into the paper disc. Wherein the diluting liquid is centrifuged (3,000 x for the supernatant after incubation g is used only the cell used after washing three times with the PBS buffer after 10 minutes). Inhibition ring size was measured drive of the entire transparent ring diameter to the diameter of the agar home. The results are shown in Table 6.
[84]
TABLE 6
Strains E. coli K88 E. coliATCC 25922 Salmonella typhimuriumKCCM 25922 Salmonella cholerasuisKCCM 10709
Lactobacillus salivarius CJLS1511 ++ ++ ++ +++
Lactobacillus salivarius KCCM 40210 + ++ + ++
10~15 mm: +, 15~20 mm: ++, 21~25 mm: +++

[85]
As can be seen from Table 6, Lactobacillus raised bariwooseu all CJLS1511 and Lactobacillus raised bariwooseu KCCM 40210 type strain was shown the antiproliferative action of the pathogenic microorganisms, the raised Lactobacillus bariwooseu CJLS1511 strain Lactobacillus raised bariwooseu KCCM 40210 standard it was confirmed that compared to the strain with high antibacterial activity.
[86]
These results demonstrate that because the effect of the cell itself, rather than the effects of the metabolites of Lactobacillus raised bariwooseu CJLS1511 is created, when it applies to livestock to inhibit harmful bacteria growth.
[87]
[88]
Experimental Example 5: Lactobacillus salivarius of CJLS1511 (KCCM11829P) digestive enzyme activity evaluation
[89]
Digestive enzyme activity tests were performed to determine whether the lactic acid bacteria with an enzyme capable of degrading carbohydrates, protein, and phosphorus.
[90]
Protease (protease) scheme in MRS agar medium in order to know whether the active milk (skim milk) 0.5 (w / v) was added to% cellulase (cellulase) MRS methyl agar cellulose in order to know whether the active (methyl cellulose) was added a 0.2 (w / v)%, a- amylase (a-amylase) to find out whether there is activity on MRS agar medium corn starch (corn starch) inserted the 0.2% (w / v) was, phytase (phytase) for the pie Tate calcium salt (Ca-phytate) to find the active was prepared medium containing 0.5 (w / v)% respectively. A smear (streaking) of the strain isolated in each medium is manufactured by the following was observed after 24 h of incubation.
[91]
a- amylase and cellulase activity by 24 hours treated with 2% of Congo Red (congo red, Sigma, USA) reagent to the culture medium after washing (washing) with a 1M sodium chloride (Nacl), then colored if it was confirmed whether or not enzymes. Further, whether or not protease and phytase activity determination was confirmed by whether or not the transparent ring, and results thereof are illustrated in Table 7.
[92]
Table 7
Strains Protease activity Phytase activity Lipase activity Cellulose activity Amylase activity
Lactobacillus salivarius CJLS1511 +++ ++ + +++ +++
Lactobacillus salivarius KCCM 40210 + + ++ ++ ++
1~10 mm: +, 11~20 mm: ++, 21~30 mm: +++

[93]
As can be seen from Table 7, the Lactobacillus salivarius CJLS1511 was found to be highly digestive enzyme activity, such as Lactobacillus salivarius KCCM 40210 protease activity compared to the type strain, pie other dehydratase activity, trehalose degrading activity, amylase activity as serul .
[94]
These results demonstrate that it is possible to improve the efficiency of Lactobacillus feed city alive applying bariwooseu CJLS1511 livestock.
[95]
[96]
Experimental Example 6: Lactobacillus salivarius of CJLS1511 (KCCM11829P) Evaluation triglyceride degradation activity
[97]
Lactobacillus raised results of testing the Bile salt hydrolase (hereinafter BSH) activity of bariwooseu CJLS1511, to produce a white precipitate in a MRS solid medium supplemented with taurodeoxycholate hydrate (hereinafter TDCA, Sigma, USA) of 2mM was confirmed that the enzymatic activity, TDCA positive control strain Lactobacillus salicylate showed a similar precipitation patterns and precipitation of bariwooseu KCCM 40210 type strain. But it was in the medium supplemented with Sodium glycodeoxycholate (hereinafter GDCA, Sigma, USA) in 2mM Lactobacillus raised for bariwooseu CJLS1511 strain but bacteria grew well, did not show the Lactobacillus raised for bariwooseu KCCM 40210 type strain precipitation, the fungus It did not grow well. The results are shown in Table 8.
[98]
Table 8
Composite bile / isolates people Lactobacillus salivarius CJLS1511 Lactobacillus salivarius KCCM 40210
TDCA O O
GDCA + -

[99]
O: precipitation, X: Does not form precipitate, +: growth, -: did not grow
[100]
[101]
As can be seen from Table 8, Lactobacillus salivarius CJLS1511 has high dapjeup resistant functionality in the conversion into a form that can degrade triglyceride in the BSH production, it can impact upon body weight gain applying them to animals it was confirmed to be a strain.
[102]
[103]
Preparation Example 1
[104]
The Lactobacillus salivarius and Lactobacillus salivarius CJLS1511 KCCM 40210 culture and smear the type strain in each loop to the MRS agar medium and cultured at 37 ℃ 48 hours to manufacture.
[105]
Then, using a heat exchanger to the culture the culture liquid was heated indirectly to a temperature of 100 ℃, cooled rapidly to 10 ℃ it at a rate of 30 ~ 100L / min. (3,000 x rapid centrifugation the cells were used in the cooled culture medium separator g by separating through 10 minutes), it raised Lactobacillus bariwooseu was prepared CJLS1511 four cells and the cells were used Lactobacillus raised bariwooseu KCCM 40210, respectively.
[106]
Preparation Example 2
[107]
The Lactobacillus salivarius and Lactobacillus salivarius CJLS1511 KCCM 40210 culture and smear the type strain in each loop to the MRS agar medium and cultured at 37 ℃ 48 hours to manufacture.
[108]
Then, using a heat exchanger to the culture the culture liquid was heated indirectly to a temperature of 100 ℃, cooled rapidly to 10 ℃ it at a rate of 30 ~ 100L / min. (3,000 x the four cells were centrifuge at rapidly cooled culture medium g by separating through 10 minutes), Lactobacillus raised bariwooseu CJLS1511 four cells and Lactobacillus save manufacturing the a bariwooseu KCCM 40210 four cells, respectively, and yeast extract here, the dextrose and sugar were mixed, respectively. After suspending the mixture in water, to give a powder by spray-drying the resultant suspension. At this time, that is, the inlet temperature was 150 ℃, the outlet temperature of the heated air was spray-dried by a 100 ℃. The amount of protective agent is, based on 100 parts by weight of the weight of the four cells, the addition amount of 20 parts by weight of the added amount of yeast extract, the amount of the index tree rose to 30 parts by weight of raw sugar was 5 parts by weight.
[109]
[110]
Respect to the four cells were prepared in Preparative example 1, the hydrophobicity and cohesiveness experiment in the manner described below, the body weight gain and feed conversion of Lactobacillus salivarius base containing CJLS1511 four cell feed and basal diet that does not contain this It was compared.
[111]
[112]
Experimental Example 7: Lactobacillus salivarius CJLS1511 ( KCCM11829P ) live cells and of dead cells hydrophobicity and cohesiveness evaluation
[113]
Lactobacillus salivarius CJLS1511 to confirm the difference between hydrophobic and cohesion of live and dead, who were evaluated for flocculation and coagulation simultaneous reaction.
[114]
Self-agglutination is Lactobacillus raised OD for bariwooseu CJLS1511 live cells and dead cells of 600 were added to 1mL of toluene in a 3mL lactic acid diluted to 0.5 was vortex for 90 seconds. After OD after removal of the aqueous layer with toluene then allowed to stand for 1 hour at 37 ℃ water bath 600 was measured.
[115]
Co-coagulation reaction is Lactobacillus raised against pathogenic microorganisms bariwooseu CJLS1511 live cells and dead cells ( E. coli K88, Salmonella typhimurium KCCM 25922, Salmonella cholerasuis mixed in KCCM 10709) in the same amount (pathogen: live cells or dead cells = 1: 1 (each 1.5 ml)) were added to 1mL of toluene and at a 3mL was vortex for 90 seconds. After OD after removal of the aqueous layer with toluene then allowed to stand for 1 hour at 37 ℃ water bath 600 was measured.
[116]
Hydrophobicity (%) is 100 x (initial OD 600 - 1 sigan OD 600 ) / initial OD 600 was calculated.
[117]
Simultaneous self-agglutination and flocculation results of live cells and dead cells was shown in the following Table 9.
[118]
Table 9
Item / pathogenic strains Lactobacillus salivarius CJLS1511
Viable Four cells
Self-agglutination 11% 23%
Co-agglutination E. coli K88 54% 64%
Salmonella typhimurium KCCM 25922 45% 65%
Salmonella cholerasuis KCCM 10709 30% 45%

[119]
As can be seen from Table 9, Lactobacillus salicylate showed self bariwooseu CJLS1511 four cells were approximately 1.5-fold higher than the live cells both flocculation and co-flocculation. More of the microorganism cells hydrophobicity and cohesiveness is influenced by characteristics and surface structure of the cell surface protein, Lactobacillus salivarius CJLS1511 four cells is different from the surface protein structure of viable cells as shown in Figure 1 is considered that the hydrophobic and aggregation properties increase. Thus, the availability of a functional strain affecting hangbyeongryeok attached to endotoxin can be expected.
[120]
[121]
Experimental Example : 8 Lactobacillus salivarius CJLS1511 ( KCCM11829P ) four the cells were compared in the feed and common basal diet comprising
[122]
Salicylate reason Lactobacillus bacteria in a basal diet for broiler bariwooseu for CJLS1511 four cells were 0.2% (w / w) a feed and a basal diet without administration of them (control) administered with, a 29 days-class, such as a normal feeding discloses the average weight (g), end the average weight (g), the average weight gain per day (g / d), average daily feed intake (g / d) and feed conversion were measured. The results are shown in Table 10.
[123]
[Table 10]
division Controls Lactobacillus salivarius CJLS1511 four cells group
Marie exam 150 150
The average start weight (g) 38.48 38.47
End the average weight (g) 1582.00a 1690.24b
Daily gain (g / d) 53.23 of a 56.96 . B
Daily feed intake (g / d) 78.76 79.48
FCR 1.48a 1.40b

[124]
a, b Average of two groups each represented by a different letter are statistically significant difference is present (P <0.05).
[125]
As shown in Table 10, taking advantage of the present invention Lactobacillus bacteria bariwooseu CJLS1511 four cells treated group showed a superior effect of both weight gain and feed conversion compared to the control group not administered them.

[126]
Claims
[Claim 1]
Lactobacillus salivarius CJLS1511 ( Lactobacillus salivarius CJLS1511) (KCCM11829P) or a four cell.
[Claim 2]
Lactobacillus salivarius CJLS1511 (KCCM11829P) or animal feed additive composition comprising a counter four cells.
[Claim 3]
The method of claim 2, wherein said animal feed additive composition containing the four cell, animal feed additive composition.
[Claim 4]
The method of claim 2, wherein the Lactobacillus salivarius CJLS1511 (KCCM11829P) or its four cells are animal feed additive composition 1g per 1.0x10 8 cfu to 1.0x10 10 , the animal feed additive composition which comprises as cfu.
[Claim 5]
Wherein the second to fourth process according to any one of the preceding claims wherein the Lactobacillus salivarius CJLS1511 (KCCM11829P) or animal feed additive composition further comprises a protective agent of its four cells.
[Claim 6]
The method of claim 5, wherein the protective agent yeast, yeast extract, monosaccharides, polysaccharides, starches, at least one member, the animal feed additive composition of seoltangryu added.
[Claim 7]
Second to claim according to any one of claims 4, wherein the animals are birds, animal feed additive composition.
[Claim 8]
Wherein the second to fourth process according to any one of the preceding claims wherein the Lactobacillus salivarius CJLS1511 (KCCM11829P) or a four cells of 0.1% to 1% by weight of the animal feed additive composition, based on weight of the animal feed.
[Claim 9]
Animal feed comprising an animal feed additive composition according to claim 8.
[Claim 10]
Lactobacillus salivarius CJLS1511 (KCCM11829P) cultured to prepare a culture solution, and by heating the culture solution at a temperature of 70 ℃ to 160 ℃, it cooled to said heated culture solution to a temperature in the range of 10 ℃ to 60 ℃, and the cooling Lactobacillus raised in a culture liquid bariwooseu CJLS1511 (KCCM11829P) raised, Lactobacillus, which comprises separating the microbial cells used bariwooseu CJLS1511 (KCCM11829P) method for producing a four cell.
[Claim 11]
11. The method of claim 10, wherein the heating is raised, that is Lactobacillus indirect heating through a heat exchanger bariwooseu CJLS1511 (KCCM11829P) method for producing a four cell.
[Claim 12]
The method of claim 10 wherein the cooling is raised, Lactobacillus is cooled at a rate of 10 ℃ / min ~ 60 ℃ / min bariwooseu CJLS1511 (KCCM11829P) method for producing a four cell.
[Claim 13]
To claim 10 according to any one of claims 12, further comprising mixing a protective agent in the separated four cells, Lactobacillus salivarius CJLS1511 (KCCM11829P) method for producing a four cell.
[Claim 14]
14. The method of claim 13, further comprising powdering the mixed four cells, Lactobacillus salivarius CJLS1511 (KCCM11829P) method for producing a four cell.
[Claim 15]
14. The method of claim 13, wherein said protective agent yeast, yeast extract, monosaccharides, polysaccharides, starches, save at least one member, of the Lactobacillus seoltangryu bariwooseu CJLS1511 (KCCM11829P) method for producing a four cell.